The brain is one of the most metabolically active organs with glucose representing the most important, but not the only, source of energy and carbon. Like all cancers, glioblastoma, the most prevalent and malignant primary brain tumor, requires a continuous source of energy and molecular resources for new cell production with a preferential use of aerobic glycolysis, recognized as the Warburg effect. Aerobic glycolysis diminishes the need for oxygen, while inefficient liberation of energy permits residual carbons to be shunted to produce cellular components. Glioblastoma is a highly lethal cancer type with almost all targeted therapeutics tested to date showing minimal to no sustained clinical benefit. The failure to achieve cure has many causes, but we and others have linked self-renewing, highly tumorigenic glioma stem cells (GSCs) to therapeutic resistance, invasion into normal tissues, and increased angiogenesis. Targeting GSCs can inhibit tumor growth and sensitize tumors to conventional therapies. In this application, two leading GSC laboratories with complementary research foci have joined forces to examine the relationship between GSCs and metabolism. Isocitrate dehydrogenase 1 (IDH1) mutations directly link transformation and metabolism in low grade gliomas and secondary glioblastoma, but these mutations are relatively rare in glioblastoma, suggesting that alternative metabolic alterations are likely present. Our two groups have interrogated GSC glucose metabolism within the cellular hierarchy (intratumoral heterogeneity) and between tumors (intertumoral heterogeneity). In preliminary studies, we found that nutrient restriction mimicking tumor conditions enriches for GSCs through preferential GSC survival and acquisition of stem- like features in differentiated cells. GSCs respond to low glucose by preferential uptake of glucose through the expression of a specialized, high affinity neuronal glucose transporter (Glut3). Glut3 enriches for GSCs and targeting Glut3 expression attenuates stem cell self-renewal and tumor growth. GSCs are not static, but rather evolve during treatment. GSCs from proneural and mesenchymal glioblastomas display differential gene expression profiles and radiation sensitivity with increased glycolytic activity in mesenchymal GSCs. Radiation induces a proneural-to-mesenchymal transition associated with activation of glycolytic metabolism and aldehyde dehydrogenase activity. As selected metabolic nodes are amenable to therapeutic targeting, we hypothesize that the Warburg effect causally contributes to glioma heterogeneity through cellular hierarchical and clonal evolution mechanisms. In this application, the first aim will determine the tumor microenvironment reprograms glucose uptake to instruct glioblastoma cellular hierarchies. As an independent yet complementary study, the second aim will determine the role of glycolytic reprogramming in the evolution of glioma stem cells to acquire therapeutic resistance. The ultimate goals of these studies are to establish a new concept by incorporating both the cellular hierarchical theory and the clonal evolution theory to better clarify the mechanism of tumor heterogeneity and to establish targeted therapies for distinct molecules that are responsible for a gain of resistance t current therapies. The proposed studies, when completed, will challenge the current research and clinical practice hurdles and will create a firm path to translation by developing a novel strategy to target the newly identified metabolic alterations in glioblastomas.